Pseudomonas aeruginosa is a motile gram-negative rod, approximately 0.5 .mu.m.times.1.5-3.0 .mu.m, with a single flagellum and occurs widely in soil, water, sewage and human intestine (See, Mol. Microbiol. 4, 1069-1075, 1990). Pseudomonas aeruginosa is a pathogenic strain causative of inveterate infections such as septicemia, generalized infection, chronic respiratory tract infection, cystic fibrosis, etc. The septicemia caused by Pseudomonas aeruginosa is a disease resulting from either the invasion of the microorganism itself or the secretion of its toxic components into the blood of patients who have lowered resistance due to surgery, laceration, trauma and the like. The presence of the toxin causes shock with high fever, reduced blood pressure, and other symptoms, which ultimately may lead to death. Furthermore, since Pseudomonas aeruginosa has been detected in urinary tract infections, interest in Pseudomonas aeruginosa has greatly increased. Accordingly, development of a medicinal agent(s) capable of effectively preventing or treating inveterate suppurative diseases, such as septicemia and urinary tract infections, caused by Pseudomonas aeruginosa is urgently needed. However, Pseudomonas aeruginosa strains are resistant to most antibiotic substances and an effective preventive or therapeutic agent for Pseudomonas aeruginosa infections has not been developed up to date. Thus, the lethality by Pseudomonas aeruginosa infections has increased over time.
Pseudomonas aeruginosa strains can be classified in various ways. One such classification system classifies the strains into seven (7) types according to Fisher immunotype See, J. Bacteriol., Vol. 98, No. 2, p. 835-836, May, 1969.! Another system is based on the 0-antigen group as proposed by Terada. Still another system is the International Antigenic Typing Scheme (IATS) classification system. In view of classification systems, the Pseudomonas aeruginosa strains most frequently occurring in Pseudomonas aeruginosa-infected patients are: 5/2a, 2c, 3, 7/3a, 3c, 1/4a, 4b, 6/5a, 5b, 4/6, 2/7a, 7b, 7c, /10a, /13, /12, /11 and 3, 7/3d, 3e types according to Fisher immunotype/O-serotype, with 3, 7, 2 and 1 types as Fisher immunotype (corresponding to 3a, 3c, 3d, 3e, 7a, 7b, 7c, 4a and 4b as O-serotype) being mainly present.
One method of treatment for Pseudomonas aeruginosa infections is to neutralizes the Pseudomonas aeruginosa toxins with antitoxins. However, the antitoxin is a therapeutic agent, which is only beneficial in patients already suffering from septicemia, and has no prophylactic effect. Further, the antitoxin recently available is very expensive and its use is limited. In addition, the most significant disadvantage accompanying the use of the antitoxin is the relatively low survival rate and the accompanying severe side effects.
One method under study to avoid the disadvantages associated with the antitoxin uses a common antigen for the prevention and treatment of Pseudomonas aeruginosa infections. Methods under study for obtaining the common antigen can be generally classified into two groups. The first method relates to the use of a common antigen separated and purified from Pseudomonas aeruginosa strains having different immunotypes as a prophylactic vaccine antigen against Pseudomonas aeruginosa infections See, Japan, J. Exp. Med. 45, 355-359, 1975!. The second method relates to the use of common antigen mass produced by utilizing a genetic engineering technique in which a gene coding for the desired antigen is isolated and inserted into a suitable vector to obtain a recombinant vector and then the suitable host is transformed with the resulting recombinant vector and cultivated to express the desired antigen.
Although development of a prophylactic vaccine using a common antigen is a very effective method in theory, at present the progress of study relating to this method indicates that this method has many problems which remain unsolved. The major disadvantage is that the common antigen cannot prevent all kinds of Pseudomonas aeruginosa infections having different immunotypes and therefore its protective efficacy is extremely low. Such low efficacy suggests that the presence of another unknown major antigen, in addition to the common antigen, may provoke an effective prophylactic effect.
Another method for treating Pseudomonas aeruginosa infections is the administration of antibiotics or chemotherapeutic agents having broad-spectrum selectivity for the Pseudomonas aeruginosa strain. However, since there are numerous Pseudomonas aeruginosa strains and they generally have a very high degree of drug resistance, many patients have succumb to Pseudomonas aeruginosa strains which cannot be effectively treated by antibiotics.
In addition, a method using therapeutic immunoglobulin has been developed. However, such immunoglobulin exhibits no or little therapeutic effect on all Pseudomonas aeruginosa infections and thus has been used only for very limited types of Pseudomonas aeruginosa infections. This is mainly due to the immunoglobulin being prepared according to a method for preparing a polyclonal antibody to a certain microorganism and therefore cannot commonly act on the numerous Pseudomonas aeruginosa strains.
Attempts have been made to develop an inexpensive therapeutic agent with mouse monoclonal antibodies See, J. Inf. Dis. 152, pp. 1290-1299, 1985! or human monoclonal antibodies See, FEMS Microbiol. Immunol. 64, pp. 263-268, 1990! against Pseudomonas aeruginosa. Here, cell lines can be selected to produce the most effective neutralizing antibodies from a cell bank with cell fusion technique and then a cell line can be used as starting material to produce the desired antibody on an industrial scale. However, this method aims at the treatment of Pseudomonas aeruginosa infection via antibody production but not at prophylactic vaccines. In addition, this method has a disadvantage that since all infections are caused by different Pseudomonas aeruginosa strains with different serological and immunological types, they cannot all be treated with only one kind of monoclonal antibody. That is, monoclonal antibody therapy cannot be effectively utilized to treat all Pseudomonas aeruginosa infected patients.
To broaden effective treatment by this method, concurrent administration of several kinds of antibodies on the basis of their investigated cross-reactivity has been developed. Here, blood is collected from Pseudomonas aeruginosa infected patients and examined to identify the serological and/or immunological type of the infecting Pseudomonas aeruginosa strains. Then monoclonal antibodies suitable to the identified type are administered to the patient. However, this method requires a lot of time and therefore is unavailable to patient whose condition necessitates immediate treatment.
In the prior art, the use of cell wall proteins separated from Pseudomonas aeruginosa strains as an antigen for a vaccine has been proposed See, Stanislavsky et al., Vaccine, Vol. 7, "Experimental studies on the protective efficacy of a Pseudomonas aeruginosa vaccine", pp. 563-565, 1989!. The Stanislavsky et al. reference describe the selection of Pseudomonas aeruginosa strains that can provide the cross-reactivity. Also disclosed are the toxicity and the protective immunogenicity of a protein polyvalent vaccine (PV) produced therefrom.
However, the method proposed in the above reference uses four kinds of general Pseudomonas aeruginosa strains, i.e. NN 170041, NN 170015, NN 868 and NN 170046, which are not attenuated and, therefore, a problem concerned with the toxicities of the Pseudomonas aeruginosa strains themselves is present.
In addition, the chemical composition of the vaccine according to the Stanislavsky et al appears in Table 1 on page 563. Table 1 clearly shows that the PV contains 0.06.+-.0.02% (w/v) of LPS. It is unclear as to whether antigenicity comes from the protein or from LPS in the Stanislavsky et al vaccine. Sawada et al pointed out that the Index of Efficacy (EI) of LPS is superior by about 40-200 times to a protein See, J. Infect. Dis., Vol. 150, No. 4, pp 570-576 (1984)!. More particularly, as described in the Abstract, the amount of LPS which elicits 50% of the protective dose (PD.sub.50) ranges 0.05-2.5 .mu.g of Ig/mouse, while that of protein (cell wall protein) ranges between 10 and 100 .mu.g of Ig/mouse. This means that about 40-200 times the smaller amount of LPS than the protein immunogen shows a similar protection against a Pseudomonas aeruginosa infection. Furthermore, even if the above amount of protein reveals PD.sub.50, much more protein immunogen (about 200 times more than the amount of LPS content) should be administrated into a subject in order to provide a 90 to 100% protective effect.
The LPS content of 0.06.+-.0.02% (w/v) in the Stanislavsky et al vaccine corresponds to about 900 to 1,800 ng per 1 mg of cell wall protein (CWP) when it is converted into weight (gram) units. In detail, when the protein content is 44.6% (w/v) and LPS is 0.06%, then the protein/LPS ratio is 743.3 (See, page 563, lines 7 to 11 of the Results). When LPS is 0.04%, the amount of LPS in 1 mg of CWP is 1/743.3.times.0.04/0.06=897 ng. When LPS is 0.08%, the amount of LPS becomes 2 times the amount, i.e., 1794 ng. This amount is enough to elicit antigenicity.
Elin et al describe that more than 300 ng of LPS content per 1 mg of cell wall 30 protein may cause toxicities such as pyrogenic symptoms and the like See, J. Infect. Dis., 128; pp. 349-352, 1973!. More specifically, the minimal pyrogenic dose is 5 ng/kg in an endotoxin from Klebsiella as described in Table 1 at page 351; this amount corresponding to 300 ng in human (body weight of 60 kg). Thus, the LPS content of ca. 900-1800 ng in the Stanislavsky et al vaccine may cause toxic side effects when it is clinically applied to human. Moreover, Edelman et al describe that the active immunization of patients with an LPS-based Pseudomonas aeruginosa vaccine was not tolerated well due to adverse reactions associated with a high endotoxin content See, page 1288, lines 2 to 5, right side column, Vaccine, Vol. 12, No. 4, pp. 1288-1294, (1994)!.
Furthermore, Stanislavsky et al show the clinical experimental results employing the same PV in another published article See, Vaccine, Vol. 9, pp 491-494, July 1991!. The PV contains 0.06.+-.0.02% (w/v) of LPS. See, page 491, lines 13-14, right side column. Table 1 on page 493 shows that 31 volunteers (26%) among 119 volunteers have experienced febrile reactions and 48 volunteers (40.3 %) experienced systemic reactions (i.e., hyperglycemia).
It is believed that, in connection with the process for preparing the vaccine, Stanislavsky et al references employ a process in which after the cell wall of the microorganism is first disrupted, the resulting mixture is then purified by centrifugation. Stanislavsky et al disclose their process in detail in a reference See, Vaccine, volume 7, pp. 562-566, December 1989!. A review of this article reveals that in the Stanislavsky et al process, the cell wall is disrupted. That is, the vaccine produced according to their process contains cytoplasmic cell components, such as, 27.2% (w/v) of nucleic acid, and 2.36% (w/v) of carbohydrates, or other components, such as 11.8% (w/v) of ash and 0.08% (w/v) of LPS in addition to 44.6% (w/v) of the target proteins.
In addition, since in preparing the above vaccine from such Pseudomonas aeruginosa strains, cell walls had to be destroyed to result in contamination of the medium with cytoplasmic substances such as nucleic acids and toxic high molecular substances which are present in cytoplasm, or lipopolysaccharides (LPS). These substances are then incorporated into the resulting vaccine as impurities increasing the care required in administering the vaccine and possibly restricting its use. Also, the vaccines prepared according to the conventional techniques contain a large amount of lipopolysaccharide, lipid-protein-carbohydrate component which may elicit antigenicity. Hitherto, there have not been known protein vaccines containing only cell wall proteins of Pseudomonas aeruginosa having molecular weight range between 10,000 and 100,000, of which antigenicity come from cell wall proteins of Pseudomonas aeruginosa strains, and thus can be desirably used without eliciting any side effects in the subject to be administrated.